Abstract: Pneumonia is the most common cause of infection-related mortality. MRSA an emerging pathogen, now accounts for more than 55% of S. aureus -related infections1. MRSA causes necrotizing pneumonia and pleural empyema. As empyema progress, fibrosis of pleural space progress and fibrothorax ultimately develop. TGF-21 is known to induce pleural fibrosis. Recently, we reported that TGF-21 activated PMC undergo EMT2. However, the exact mechanisms that cause fibrosis in pleural empyema are unknown and the precise role of PMC in the pathogenesis of pleural fibrosis remains unclear. We propose to study the mechanisms of pleural fibrosis in a HO-1 knock out mouse model because defective HO-1 expression was associated with increased susceptibility to bacterial pneumonia in an older population3. Furthermore, HO-1 deficiency is known to promote EMT in renal fibrosis4 and HO-1 induction prevented pulmonary fibrosis in animal models5. MicroRNA (miR) are a major class of gene expression regulators linked to many biological functions including cell proliferation and morphogenesis. In preliminary studies, in MRSA infected HO-1 -/- PMC, we found an exaggerated expression of miR-26a and increased EMT in vitro. Additionally, HO-1 absence contributed to increased pleural fibrosis in our murine model of MRSA empyema in vivo. A search in MicroCosm Targets data base revealed that miR-26a targets E-cadherin (cell junction protein) which maintains epithelial phenotype and membrane integrity. However, the mechanisms whereby this occurs in pleural empyema are unclear. We have a novel and well established murine model of pleural empyema to evaluate the mechanisms of pleural fibrosis in vivo. In addition, we have a unique expertise in PMC isolation from mice7 that will allow us to dissect the mechanisms of EMT in primary cultures of PMC in vitro. Based on our preliminary studies we hypothesized that in MRSA empyema, PMC undergo EMT and HO-1 plays a critical role in regulation of miR-26a expression and SMAD-2 signaling in pleural fibrosis. Using our novel murine model we will determine: 1) If HO-1 regulates PF via miR-26a expression in a murine model of MRSA empyema. 2) If miR-26a regulate PMC-EMT in MRSA empyema. 3) If HO-1 regulates SMAD-2 signaling and modulate EMT in MRSA infected PMC in vitro. This study is innovative because: a) it is the first study to assess if PMC in the presence of infection/inflammation undergo EMT and contributes to fibrosis in pleural empyema; b) the significance of HO- 1, a cytoprotective molecule, in pleural fibrosis will be assessed; and c) we will investigate the role of a novel micro RNA, the miR-26a in pleural fibrosis in empyema; d) we will use a unique approach to deliver the therapeutics directly to the site of fibrosis in the pleura and prevent unnecessary exposure of unaffected tissues. This project is highly significant because, the studies are designed to gain an understanding of the mechanisms associated with PMC EMT and fibrosis during pleural empyema. The data obtained from this project will allow us to assess the potential benefits of developing interventions that target pleural fibrosis via two distinct approaches; 1) via HO-1 induction by pharmacological means to prevent fibrosis in pleural empyema; 2) via targeting miR-26a by intrapleural gene therapy to prevent fibrosis in pleural empyema. Currently there are no effective treatments for pleural fibrosis therefore the findings from these studies will begin to build the knowledge necessary to move towards development of potential therapeutics for the pleural empyema Veteran patients. PUBLIC HEALTH RELEVANCE: MRSA is an emerging respiratory pathogen in both community-associated and hospital-associated infections that often leads to highly lethal empyema and pleural fibrosis. In recent years MRSA infections have become common in outpatients of army community hospitals and among military recruits, in nursing home care units of VAMC and in Operation Iraqi Freedom troop population. Furthermore, MRSA infections are a major and growing cause of hospital-associated pneumonia in patients with trauma, surgery and spinal cord injury. The clinical management of empyema is associated with prolonged hospitalization, and instrumentation of the pleural space with significant patient discomfort. Here we propose two distinct treatment approaches; 1) via HO-1 induction by pharmacological means to prevent fibrosis in pleural empyema; and 2) via targeting miR-26a by intrapleural gene therapy to prevent fibrosis in pleural empyema. Currently there are no effective treatments for pleural fibrosis therefore the findings from these studies will move towards development of potential therapeutics against empyema and help to better manage empyema patients.